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. 2021 Apr 13;12(1):2211.
doi: 10.1038/s41467-021-22400-z.

Conformational interconversion of MLKL and disengagement from RIPK3 precede cell death by necroptosis

Sarah E Garnish #  1   2 , Yanxiang Meng #  1   2 , Akiko Koide #  3   4 , Jarrod J Sandow  1   2 , Eric Denbaum  3 , Annette V Jacobsen  1   2 , Wayland Yeung  5 , Andre L Samson  1   2 , Christopher R Horne  1   2 , Cheree Fitzgibbon  1 , Samuel N Young  1 , Phoebe P C Smith  1 , Andrew I Webb  1   2 , Emma J Petrie  1   2 , Joanne M Hildebrand  1   2 , Natarajan Kannan  5   6 , Peter E Czabotar  1   2 , Shohei Koide  7   8 , James M Murphy  9   10
Affiliations

Conformational interconversion of MLKL and disengagement from RIPK3 precede cell death by necroptosis

Sarah E Garnish et al. Nat Commun. .

Abstract

Phosphorylation of the MLKL pseudokinase by the RIPK3 kinase leads to MLKL oligomerization, translocation to, and permeabilization of, the plasma membrane to induce necroptotic cell death. The precise choreography of MLKL activation remains incompletely understood. Here, we report Monobodies, synthetic binding proteins, that bind the pseudokinase domain of MLKL within human cells and their crystal structures in complex with the human MLKL pseudokinase domain. While Monobody-32 constitutively binds the MLKL hinge region, Monobody-27 binds MLKL via an epitope that overlaps the RIPK3 binding site and is only exposed after phosphorylated MLKL disengages from RIPK3 following necroptotic stimulation. The crystal structures identified two distinct conformations of the MLKL pseudokinase domain, supporting the idea that a conformational transition accompanies MLKL disengagement from RIPK3. These studies provide further evidence that MLKL undergoes a large conformational change upon activation, and identify MLKL disengagement from RIPK3 as a key regulatory step in the necroptosis pathway.

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Conflict of interest statement

S.E.G., A.L.S., C.F., S.N.Y., E.J.P., J.M.H., P.E.C., and J.M.M. contribute, or have contributed, to a project developing necroptosis inhibitors in collaboration with Anaxis Pharma Pty Ltd. A.K. and S.K. are listed as inventors on issued and pending patents on the Monobody technology filed by The University of Chicago (US Patent 9512199 B2 and related pending applications). The other authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Mb27 binds activated human MLKL.
a Wild-type (WT) HT29 cells expressing Mb27 or Mb32 following doxycycline (Dox) induction were untreated (UT) or treated with the necroptotic stimulus, TSI (TNF; Smac mimetic, Compound A; and pan-Caspase inhibitor, IDN-6556), for 3 h and monobodies immunoprecipitated via their N-terminal FLAG-tag. Mb32 coimmunoprecipitated MLKL in both presence and absence of TSI stimulation, whilst Mb27 coimmunoprecipitated MLKL only in the presence of TSI. b Mb27 immunoprecipitated MLKL from lysates of RIPK3−/− HT29 cells in the absence and presence of necroptotic stimulation (TSI, 3 h). c Wild-type HT29 cells were treated with a necroptotic stimulus (TSI) over a 3 h timecourse, and Mb27 immunoprecipitation of MLKL examined. Mb27 co-immunoprecipitation of MLKL followed the emergence of RIPK3-mediated MLKL S358 phosphorylation (pMLKL) 1.5 h post-necroptotic stimulation. All images and blots are representative of at least two independent experiments. d FLAG-immunoprecipitates of Mb27 or Mb32 expressed (+Dox) in wild-type HT29 cells ±7.5 h TSI treatment revealed the presence of pRIPK3 bound to MLKL, with Mb27 immunoprecipitating MLKL only after TSI-stimulation. A representative of 4 independent experiments is shown. e FLAG-human RIPK3 expressed (+Dox) in RIPK3−/− HT29 cells coimmunoprecipitates MLKL in the presence or absence of TSI-stimulation, but not pMLKL post-TSI treatment. Lysates and IP samples were run on the same gel and subjected to the same exposure. A representative of independent duplicates is shown in each of (c, e). f Recombinant His6-human MLKL pseudokinase domain and RIPK3 kinase domain complex expressed and purified from insect cells was untreated (UT) or incubated with combinations of Mg2+, ATP or AMPPNP, the complex trapped by Ni-NTA chromatography and the eluates probed by immunoblot for RIPK3 and MLKL. Only ATP/Mg2+ induced dissociation of RIPK3 and MLKL. Triplicates are shown for each condition.
Fig. 2
Fig. 2. Mb27 and Mb32 bind distinct human MLKL epitopes.
a, b, d, e Transverse views of the monobody:human MLKL pseudokinase domain co-crystal structures. a, b Mb27 (β-sheets shown in light and dark blue) binds the ATP-binding cleft of the pseudokinase domain (gray; αC helix, yellow), atop the activation loop (residues 351-372; green), via its BC (raspberry), DE (salmon), and FG (light pink) loops. c Mb27 residues within 4.5 Å of MLKL are shown as sticks (colored as above). d, e Mb32 (β-sheets shown in marine and cyan) binds the hinge region of human MLKL pseudokinase domain (gray; αC helix, yellow; activation loop, green) principally via the Mb32 elongated FG loop, the CD loop and the D, G and F β-strands. f Mb32 residues within 4.5 Å of MLKL are shown as sticks (colored as above).
Fig. 3
Fig. 3. Human MLKL exists in two distinct conformations.
a–f A comparison of the unliganded human MLKL pseudokinase domain structure (a; PDB accession 4MWI), the human MLKL pseudokinase domain from the Mb27 (b) and Mb32 (c) complexes and the rat (d; PDB accession 6VBZ), horse (e; PDB accession 6VC0); and mouse (f; PDB accession 4BTF) MLKL pseudokinase domain structures. Residues of the R-spine are shown as sticks in (b, c). Zoomed panels show the human MLKL K230–E250 (or equivalent residues in ortholog structures) salt bridge between the β3 strand ATP-binding lysine and the glutamate in the αC helix (yellow). A glutamine from the activation loop (green) helix hydrogen bonds with the β3 strand ATP-binding lysine in the open kinase conformation in mouse MLKL and the Mb32-bound human MLKL structure. g Overlaid Cα backbone traces of the previously-reported human MLKL pseudokinase domain structure (PDB 4MWI; gray) and the Mb27- (blue) and Mb32 (red)-bound MLKL conformers reported herein. h Model illustrating the stable hydrogen bond between T246 on the αC helix and pS358 on the activation loop helix of human MLKL observed in molecular dynamics simulations. i Molecular dynamics simulations of the human MLKL pseudokinase domain open conformer containing the activation loop helix (from the Mb32 complex structure) at the microsecond timescale. Dephosphorylated MLKL is shown on the left and phosphorylated MLKL is shown on the right. Phosphorylated residues pT357 and pS358 are marked with oranges triangles below the sequence of pMLKL. Helices are marked in red and β-strands are marked in blue. Five independent replicates are shown. j The transition from open, Mb32-bound conformation to the closed conformation (from PDB, 4MWI; top) was simulated and the two free energy surfaces along the reaction coordinate calculated as a function of the K230–E250 salt-bridge distance (lower). The ΔG was calculated as the difference between local free energy minima before and after 10 Å. Shown on the right, ΔΔG = −4.02 kcal/mol indicates the change in ΔG upon phosphorylation. A negative value suggests that phosphorylation stabilizes the closed, active-like conformation.
Fig. 4
Fig. 4. Mutation of MLKL R292 and T374 in the Mb27 binding epitope inhibits necroptosis.
a Sequence alignment of human and mouse MLKL pseudokinase domains. Residues of mouse MLKL that interact with RIPK3 in the MLKL:RIPK3 complex structure (PDB accession 4M69; as predicted by PDBePISA) in red text; key structural elements in teal; activation loop residues phosphorylated by RIPK3 in green. b The mouse MLKL pseudokinase domain (cyan):RIPK3 kinase domain (purple) co-crystal structure (PDB accession 4M69) with non-conserved residues in human MLKL and RIPK3 in red and yellow, respectively. c, d The human MLKL:Mb27 structure was superimposed on the mouse MLKL:RIPK3 complex structure (PDB 4M69), with Mb27-proximal residues identified for characterization in e shown as sticks (c). d The Mb27-binding interface on the human MLKL pseudokinase (cyan surface), residues within 4.5 Å of the superimposed mouse RIPK3 kinase domain (PDB 4M69) (purple surface) and overlapped residues in the Mb27- and mouse RIPK3-epitopes, V371, K372, S373, and S417 (orange surface) are shown. The αC helix (yellow), and previously implicated RIPK3-interacting regions of human MLKL, the activation loop (green) and F386 (blue sticks),, and T374 implicated herein, are highlighted. e Evaluation of necroptotic signaling by wild-type and Mb27 binding epitope mutants of full-length human MLKL in MLKL−/− HT29 cells. Wild-type or mutant human MLKL expression was induced with doxycycline (Dox) and cell death was measured by SYTOX Green uptake (1/mm2) quantified using IncuCyte S3 live cell imaging in the presence or absence of a necroptotic stimulus (TNF, Smac mimetic, IDN-6556; TSI) for 20 h. Two independent cell lines were generated for WT, S239A, Q236A, and one for other MLKL mutants; WT lines were assayed in n = 5 independent experiments. TSEE, R333A and one independent Q236A line were assayed in n = 4 independent experiments; other mutants in n = 3 independent experiments. Data are plotted as mean ± SEM. f Alanine substitution of MLKL R292 likely disrupts key interactions with neighboring residues. g Wild-type and R292A, but not T374D, human MLKL expressed in MLKL−/− HT29 cells were phosphorylated in response to 3 h TSI treatment. Data are representative of duplicate independent experiments.
Fig. 5
Fig. 5. Summary of regulatory checkpoints in human MLKL activation.
Necroptosis is initiated by ligand binding to cell surface death receptors, such as TNF receptor 1, when the IAP E3 ubiquitin ligases and Caspase-8 are depleted or inhibited. Immunoprecipitation using Mb32 identified MLKL to exist in complex with RIPK3 prior to initiation of necroptosis. The RIPK3:MLKL complex is recruited to a RIPK1-nucleated platform (the necrosome) following induction of necroptosis, where RIPK3 is autophosphorylated and MLKL phosphorylated by RIPK3. Subsequently, oligomeric phosphorylated MLKL (pMLKL) disengages from RIPK3, which permits its recognition by Mb27. Phospho-MLKL oligomers are subsequently trafficked to the plasma membrane via the Golgi-/actin-/microtubule-trafficking machinery. pMLKL accumulates at the plasma membrane into higher order hotspots, and when a threshold amount of MLKL is surpassed, MLKL ruptures the plasma membrane to induce cell death and release of pro-inflammatory DAMPs. Monobody-27 and Monobody-32 are depicted in light and dark purple colors, respectively; dashed lines represent the interactions observed for Mb27 and Mb32 in HT29 cells. The skull and crossbones image (Mycomorphbox_Deadly.png; artist, Sven Manguard) was sourced under a Creative Commons Attribution-Share Alike 4.0 license.
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